Hyaluronic acid (HA) or hyaluronan is a glycosaminoglucan consisting of repeating disaccharides of alternating D-glucuronic acid and N-acetylglucosamine molecules. These molecules are joined by a β (1,3)-D linkage while the glucosamine to glucuronic acid linkage is β (1,4)-D.
There are several sources of hyaluronic acid and its molecular weight varies considerably depending on the source. The HA found in synovial fluid has a molecular weight of about 1 to 8 million, that in human umbilical cord has a molecular weight around 3.6-4.5 million and HA in rooster combs may reach very high values, for instance up to 12-14 million, or even higher. The chemical composition of hyaluronic acid is the same regardless of its source and since it is non-immunogenic it has found several applications in medicine (Brimacombe and Webber (1964)). The effectiveness of HA is a result of a unique combination of elastic and viscous properties, which are correlated to the molecular weight. Therefore, there was early an interest in obtaining as high molecular weights as possible.
Accordingly, the literature contains numerous examples of very high values of the molecular weight of HA but these values very often refer to the source material. It should be noticed, however, that since the HA as produced in biological systems like rooster combs, is associated with proteins and other glycosaminoglycans, for example chondroitin sulphate, it has to be extensively purified. Even if very sophisticated methods for purification and sterilization have been developed it is inevitable that the molecular weight decreases during these steps and the final product in most cases has much lower molecular weight.
The major HA product on the market today is Healon® (Pharmacia AB, Uppsala, Sweden) which has a molecular weight around 3.5 million. This product is prepared from rooster combs according to a method based on the disclosure of U.S. Pat. No. 4,141,973. From the same source is prepared a HA product with a molecular weight around 5 million, Healon® GV (Pharmacia AB). These molecular weights refer to the sterilized products and this means that the product before the sterilization step must have molecular weights around 5 and 7 million, respectively.
There are very few high molecular weight HA products on the market, in spite of the well-documented usefulness of HA in several medical indications, for instance in ophthalmology. One reason for this is probably the complex purification procedures required in order to obtain a pure product from the sources mentioned above, especially rooster combs without too much degradation of the molecular chains. Therefore, there is a need for alternative sources or production systems which are well controlled and which allow a simplified purification procedure.
Numerous articles and patent applications have been published which relate to the production of HA in various bacterial systems. The use of bacteria for biotechnological production of HA has been advocated for several reasons, technical, economical as well as ethical. The production by Streptococcus spp. has been known for more than 50 years and most of the systems disclosed seem to refer to group A and C streptococci, for instance encapsulated strains of Streptococcus pyogenes (group A), which is a human pathogen (Kendall et al (1937)), and Streptococus equi and Streptococcus equisimilis (group C), which are animal pathogens. The synthesis of hyaluronic acid as the major capsular polysaccharide in these pathogens is a way to evade host defenses (Roberts et al (1989)).
The biochemistry of HA synthesis in bacteria involves the action of two, so far known, genes, has A coding for synthase, which is an integral membrane protein and has B coding for UDP-glucose dehydrogenase, which converts UDP glucose to UDP-glucuronic acid. In addition, UDP-glucose needs to be converted to UDP-N-acetyl glucoseamine, which is required for cell wall biosynthesis (see Dougherty and van de Rijn (1992, 1993) and de Angelis et al (1993)). The control of the synthesis, for instance what initiates and terminates HA synthesis, is much less known. However, the stoichiometry of the synthesis provides some guidelines for composition of feed and medium.
The efforts with regard to the development of bacteria-based HA production systems have been focused on the selection of bacteria and suitable culture media. It was early evident that capsulated wildtype strains did not release HA of a molecular weight higher than about 5 million into the fermentation broth, though there were indications in the literature that the actual molecular weight in the capsule might be somewhat higher, see van de Rijn (1983). However, as judged from the literature, including patents and commercially available samples, the molecular weight of bacteria-produced HA is far below that at present produced from rooster combs (see above). It should further be noticed that there often is a very clear difference between high molecular weight values indicated in the literature, which express a desired result, and the values actually obtained.
The highest values obtained in bacteria systems seems to be around 4 million, see for instance U.S. Pat. No. 4,784,990 (Bio-Technology General)—HA of 2-3.5 million; WO 92/08799 (Fermentech)—HA of 1-3 million, JP 2-058502 (Chisso Corp)—HA of 2-3 million, JP 63-129991 and JP 63-028398 (Denki Kagaku Kogyo KK)—HA of 2-4 million, and EP 144019 (Miles Laboratories, Mobay Chemical Corp)—HA of 2-4 million.
It should further be notified that the values given above refer to HA products which have not been sterilized and it's therefore clear that these materials can not be used for the manufacture of HA products which after sterilization have molecular weights comparable to the Healon® products mentioned above.
All strains of streptococci are aerotolerant anaerobes, that is they are able to grow in the presence of oxygen but they don't use oxygen as electron acceptor. Accordingly, the discussion or speculation in prior art articles and patents regarding the importance of air doesn't seem to address any parameter of crucial importance for HA production.
Suitable media and conditions for production of HA are discussed in most of the papers related to the production, and additional examples of patents or patent applications in this field include JP 63-141594 and JP 63-123392 (Denki Kagaku Kogyo KK) as well as U.S. Pat. No. 4,897,349 (MedChem Products Inc.).